Nucleotide sequences mediating male fertility and method of using same

ABSTRACT

Nucleotide sequences mediating male fertility in plants are described, with DNA molecule and amino acid sequences set forth. Promoter sequences and their essential regions are also identified. The nucleotide sequences are useful in mediating male fertility in plants.

This application is a continuation-in-part of previously filed andapplication U.S. Ser. No. 10/412,000 filed Apr. 11, 2003, now U.S. Pat.No. 7,151,205 which is a continuation of previously filed applicationU.S. Ser. No. 09/670,153, filed Sep. 26, 2000, now abandoned, both ofwhich are incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Development of hybrid plant breeding has made possible considerableadvances in quality and quantity of crops produced. Increased yield andcombination of desirable characteristics, such as resistance to diseaseand insects, heat and drought tolerance, along with variations in plantcomposition are all possible because of hybridization procedures. Theseprocedures frequently rely heavily on providing for a male parentcontributing pollen to a female parent to produce the resulting hybrid.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinating if pollenfrom one flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant.

In Brassica, the plant is normally self sterile and can only becross-pollinated. In self-pollinating species, such as soybeans andcotton, the male and female plants are anatomically juxtaposed. Duringnatural pollination, the male reproductive organs of a given flowerpollinate the female reproductive organs of the same flower.

Maize plants (Zea mays L.) present a unique situation in that they canbe bred by both self-pollination and cross-pollination techniques. Maizehas male flowers, located on the tassel, and female flowers, located onthe ear, on the same plant. It can self or cross pollinate. Naturalpollination occurs in maize when wind blows pollen from the tassels tothe silks that protrude from the tops of the incipient ears.

A reliable method of controlling fertility in plants would offer theopportunity for improved plant breeding. This is especially true fordevelopment of maize hybrids, which relies upon some sort of malesterility system and where a female sterility system would reduceproduction costs.

The development of maize hybrids requires the development of homozygousinbred lines, the crossing of these lines, and the evaluation of thecrosses. Pedigree breeding and recurrent selection are two of thebreeding methods used to develop inbred lines from populations. Breedingprograms combine desirable traits from two or more inbred lines orvarious broad-based sources into breeding pools from which new inbredlines are developed by selfing and selection of desired phenotypes. Ahybrid maize variety is the cross of two such inbred lines, each ofwhich may have one or more desirable characteristics lacked by the otheror which complement the other. The new inbreds are crossed with otherinbred lines and the hybrids from these crosses are evaluated todetermine which have commercial potential. The hybrid progeny of thefirst generation is designated F₁. In the development of hybrids onlythe F₁ hybrid plants are sought. The F₁ hybrid is more vigorous than itsinbred parents. This hybrid vigor, or heterosis, can be manifested inmany ways, including increased vegetative growth and increased yield.

Hybrid maize seed can be produced by a male sterility systemincorporating manual detasseling. To produce hybrid seed, the maletassel is removed from the growing female inbred parent, which can beplanted in various alternating row patterns with the male inbred parent.Consequently, providing that there is sufficient isolation from sourcesof foreign maize pollen, the ears of the female inbred will befertilized only with pollen from the male inbred. The resulting seed istherefore hybrid (F₁) and will form hybrid plants.

Environmental variation in plant development can result in plantstasseling after manual detasseling of the female parent is completed.Or, a detasseler might not completely remove the tassel of a femaleinbred plant. In any event, the result is that the female plant willsuccessfully shed pollen and some female plants will be self-pollinated.This will result in seed of the female inbred being harvested along withthe hybrid seed which is normally produced. Female inbred seed is not asproductive as F₁ seed. In addition, the presence of female inbred seedcan represent a germplasm security risk for the company producing thehybrid.

Alternatively, the female inbred can be mechanically detasseled bymachine. Mechanical detasseling is approximately as reliable as handdetasseling, but is faster and less costly. However, most detasselingmachines produce more damage to the plants than hand detasseling. Thus,no form of detasseling is presently entirely satisfactory, and a needcontinues to exist for alternatives which further reduce productioncosts and to eliminate self-pollination of the female parent in theproduction of hybrid seed.

A reliable system of genetic male sterility would provide advantages.The laborious detasseling process can be avoided in some genotypes byusing cytoplasmic male-sterile (CMS) inbreds. In the absence of afertility restorer gene, plants of a CMS inbred are male sterile as aresult of factors resulting from the cytoplasmic, as opposed to thenuclear, genome. Thus, this characteristic is inherited exclusivelythrough the female parent in maize plants, since only the femaleprovides cytoplasm to the fertilized seed. CMS plants are fertilizedwith pollen from another inbred that is not male-sterile. Pollen fromthe second inbred may or may not contribute genes that make the hybridplants male-fertile. Usually seed from detasseled normal maize and CMSproduced seed of the same hybrid must be blended to insure that adequatepollen loads are available for fertilization when the hybrid plants aregrown and to insure cytoplasmic diversity.

There can be other drawbacks to CMS. One is an historically observedassociation of a specific variant of CMS with susceptibility to certaincrop diseases. This problem has discouraged widespread use of that CMSvariant in producing hybrid maize and has had a negative impact on theuse of CMS in maize in general.

One type of genetic sterility is disclosed in U.S. Pat. Nos. 4,654,465and 4,727,219 to Brar, et al. However, this form of genetic malesterility requires maintenance of multiple mutant genes at separatelocations within the genome and requires a complex marker system totrack the genes and make use of the system convenient. Patterson alsodescribed a genic system of chromosomal translocations which can beeffective, but which are complicated. (See, U.S. Pat. Nos. 3,861,709 and3,710,511.)

Many other attempts have been made to improve on these drawbacks. Forexample, Fabijanski, et al., developed several methods of causing malesterility in plants (see EPO 89/3010153.8 publication no. 329,308 andPCT application PCT/CA90/00037 published as WO 90/08828). One methodincludes delivering into the plant a gene encoding a cytotoxic substanceassociated with a male tissue specific promoter. Another involves anantisense system in which a gene critical to fertility is identified andan antisense to the gene inserted in the plant. Mariani, et al. alsoshows several cytotoxic antisense systems. See EP 89/401, 194. Stillother systems use “repressor” genes which inhibit the expression ofanother gene critical to male sterility. PCT/GB90/00102, published as WO90/08829.

A still further improvement of this system is one described at U.S. Pat.No. 5,478,369 (incorporated herein by reference) in which a method ofimparting controllable male sterility is achieved by silencing a genenative to the plant that is critical for male fertility and replacingthe native DNA with the gene critical to male fertility linked to aninducible promoter controlling expression of the gene. The plant is thusconstitutively sterile, becoming fertile only when the promoter isinduced and its attached male fertility gene is expressed.

As noted, an essential aspect of much of the work underway with malesterility systems is the identification of genes impacting malefertility.

Such a gene can be used in a variety of systems to control malefertility including those described herein. Previously, a male fertilitygene has been identified in Arabidopsis thaliana and used to produce amale sterile plant. Aarts, et al., “Transposon Tagging of a MaleSterility Gene in Arabidopsis”, Nature, 363:715-717 (Jun. 24, 1993).U.S. Pat. No. 5,478,369 discloses therein one such gene impacting malefertility. In the present invention the inventors provide novel DNAmolecules and the amino acid sequence encoded that are critical to malefertility in plants. These can be used in any of the systems wherecontrol of fertility is useful, including those described above.

Thus, one object of the invention is to provide a nucleic acid sequence,the expression of which is critical to male fertility in plants.

Another object of the invention is to provide a DNA molecule encoding anamino acid sequence, the expression of which is critical to malefertility in plants.

Yet another object of the invention is to provide a promoter of suchnucleotide sequence and its essential sequences.

A further object of the invention is to provide a method of using suchDNA molecules to mediate male fertility in plants.

Further objects of the invention will become apparent in the descriptionand claims that follow.

SUMMARY OF THE INVENTION

This invention relates to nucleic acid sequences, and, specifically, DNAmolecules and the amino acid encoded by the DNA molecules, which arecritical to male fertility. A promoter of the DNA is identified, as wellas its essential sequences. It also relates to use of such DNA moleculesto mediate fertility in plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a locus map of the male fertility gene Ms26.

FIG. 2A is a Southern blot of the ms26-m2::Mu8 family hybridized with aMu8 probe;

FIG. 2B is a Southern blot of the ms26-m2::Mu8 family hybridized with aPstI fragment isolated from the ms26 clone.

FIG. 3. is a Northern Blot analysis gel hybridized with a PstI fragmentisolated from the Ms26 gene.

FIG. 4A-4D is the sequence of Ms26 (The cDNA is SEQ ID NO: 1, theprotein is SEQ ID NOS: 2 and 34)

FIG. 5A-D is a comparison of the genomic Ms26 sequence (Residues1051-3326 of SEQ ID NO: 7) with the cDNA of Ms26 (SEQ ID NO: 1).

FIG. 6A is a Northern analysis gel showing expression in various planttissues and FIG. 6B is a gel showing expression stages ofmicrosporogenesis

FIG. 7 is the full length promoter of Ms26 (SEQ ID NO: 5)

FIG. 8 is a bar graph showing luciferase activity after deletions ofselect regions of the Ms26 promoter.

FIG. 9 shows essential regions of the Ms26 promoter (SEQ ID NO: 6).

FIG. 10 is a bar graph showing luciferase activity after substitution byrestriction site linker scanning of select small (9-10 bp) regions ofthe Ms26 essential promoter fragment.

FIG. 11A and 11B is a comparison of the nucleotide sequence (SEQ ID NO:3) from the Ms26 orthologue from a sorghum panicle and Ms26 maize cDNA(Residues 201-750 of SEQ ID NO: 1), and the sorghum protein sequence(SEQ ID NO: 4) and Ms26 maize protein (Residues 87-244 of SEQ ID NO; 2).

FIG. 12 is a representation of the mapping of the male sterility genems26.

FIG. 13 shows a sequence comparison of the region of excision of thems26-ref allele (SEQ ID NO: 8) with wild-type Ms26 (SEQ ID NO: 9).

FIG. 14A shows a translated protein sequence alignment between regionsof the CYP704B1, a P450 gene (SEQ ID NO: 12) and Ms26 (SEQ ID NO: 13);FIG. 14B shows the phylogenetic tree analysis of select P450 genes.

FIG. 15 demonstrates the heme binding domain frame shift, showing thetranslated sequence alignment of regions of the Ms26 cDNA (SEQ ID NOS:14 and 28-29), the genomic regions of exon 5 in fertile plants (SEQ IDNOS: 15 and 30-31) and sterile plants (SEQ ID NOS: 16 and 32-33).

FIG. 16 shows alignment of the Ms26 promoter of corn (Residues 650-1089of SEQ ID NO: 5), sorghum (SEQ ID NO: 19) and rice (SEQ ID NO: 20).

FIG. 17 shows alignment of the maize Ms26 protein (SEQ ID NO: 21); riceMs26 protein (SEQ ID NO: 18) and sorghum Ms26 protein (SEQ ID NO: 22)along with a consensus sequence (SEQ ID NO: 35).

DISCLOSURE OF THE INVENTION

All references referred to are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated therein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

Genetic male sterility results from a mutation, suppression, or otherimpact to one of the genes critical to a specific step inmicrosporogenesis, the term applied to the entire process of pollenformulation. These genes can be collectively referred to as malefertility genes (or, alternatively, male sterility genes). There aremany steps in the overall pathway where gene function impacts fertility.This seems aptly supported by the frequency of genetic male sterility inmaize. New alleles of male sterility mutants are uncovered in materialsthat range from elite inbreds to unadapted populations. To date,published genetic male sterility research has been mostly descriptive.Some efforts have been made to establish the mechanism of sterility inmaize, but few have been satisfactory. This should not be surprisinggiven the number of genes that have been identified as being responsiblefor male sterility. One mechanism is unlikely to apply to all mutations.

At U.S. Pat. No. 5,478,369 there is described a method by which a malesterility gene was tagged and cloned on maize chromosome 9. Previously,there has been described a male sterility gene on chromosome 9, ms2,which has never been cloned and sequenced. It is not allelic to the genereferred to in the '369 patent. See Albertsen, M. and Phillips, R. L.,“Developmental Cytology of 13 Genetic Male Sterile Loci in Maize”Canadian Journal of Genetics & Cytology 23:195-208 (January 1981). Theonly fertility gene cloned before that had been the Arabadopsis genedescribed at Aarts, et al., supra.

Thus the invention includes using the sequences shown herein it impactsmale fertility in a plant, that is, to control male fertility bymanipulation of the genome using the genes of the invention. By way ofexample, without limitation, any of the methods described supra can beused with the sequence of the invention such as introducing a mutantsequence into a plant to cause sterility, causing mutation to the nativesequence, introducing an antisense of the sequence into the plant,linking it with other sequences to control its expression, or any one ofa myriad of processes available to one skilled in the art to impact malefertility in a plant.

The Ms26 gene described herein is located on maize chromosome 1 and itsdominant allele is critical to male fertility. The locus map isrepresented at FIG. 1. It can be used in the systems described above,and other systems impacting male fertility.

The maize family cosegregating for sterility was named ms*-SBMu200 andwas found to have an approximately 5.5 Kb EcoRI fragment that hybridizedwith a Mu8 probe (2A). A genomic clone from the family was isolatedwhich contained a Mu8 transposon. A probe made from DNA bordering thetransposon was found to hybridize to the same ˜5.5 Kb EcoR1 fragment(2B). This probe was used to isolate cDNA clones from a tassel cDNAlibrary. The cDNA is 1906 bp, and the Mu insertion occurred in exon 1 ofthe gene. This probe was also used to map the mutation in an RFLPmapping population. The mutant mapped to the short arm of chromosome 1,near Ms26. Allelism crosses between ms26-ref and ms*-SBMu200 showed thatthese were allelic, indicating that the mutations occurred in the samegene. The ms*-SBMu200 allele was renamed ms26-m2::Mu8. Two additionalalleles for the Ms26 gene were cloned, one containing a Mutator elementin the second exon, named ms26-m3::Mu*, and one containing an unknowntransposon in the fifth exon from the ms26-ref allele. SEQ ID NO: 7(discussed further below) represents the genomic nucleotide sequence.Expression patterns, as determined by Northern analysis, show tasselspecificity with peak expression at about the quartet to quartet releasestages of microsporogenesis.

Further, it will be evident to one skilled in the art that variations,mutations, derivations including fragments smaller than the entiresequence set forth may be used which retain the male sterilitycontrolling properties of the gene. One of ordinary skill in the art canreadily assess the variant or fragment by introduction into plantshomozygous for a stable male sterile allele of Ms26, followed byobservation of the plant's male tissue development.

The sequences of the invention may be isolated from any plant,including, but not limited to corn (Zea mays), canola (Brassica napus,Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), millet (Panicum spp.), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), oats (Avena sativa), barley (Hordeum vulgare), vegetables,ornamentals, and conifers. Preferably, plants include corn, soybean,sunflower, safflower, canola, wheat, barley, rye, alfalfa, rice, cottonand sorghum.

Sequences from other plants may be isolated according to well-knowntechniques based on their sequence homology to the homologous codingregion of the coding sequences set forth herein. In these techniques,all or part of the known coding sequence is used as a probe whichselectively hybridizes to other sequences present in a population ofcloned genomic DNA fragments (i.e. genomic libraries) from a chosenorganism. Methods are readily available in the art for the hybridizationof nucleic acid sequences. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, N.Y. (1993); and Current Protocolsin Molecular Biology, Chapter 2, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

Thus the invention also includes those nucleotide sequences whichselectively hybridize to the Ms26 nucleotide sequences under stringentconditions. In referring to a sequence that “selectively hybridizes”with Ms26, the term includes reference to hybridization, under stringenthybridization conditions, of a nucleic acid sequence to the specifiednucleic acid target sequence to a detectably greater degree (e.g., atleast 2-fold over background) than its hybridization to non-targetnucleic acid.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare target-sequence-dependent and will differ depending on the structureof the polynucleotide. By controlling the stringency of thehybridization and/or washing conditions, target sequences can beidentified which are 100% complementary to a probe (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, probes of this type are in arange of about 1000 nucleotides in length to about 250 nucleotides inlength.

An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). See also Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

In general, sequences that correspond to the nucleotide sequences of thepresent invention and hybridize to the nucleotide sequence disclosedherein will be at least 50% homologous, 70% homologous, and even 85%homologous or more with the disclosed sequence. That is, the sequencesimilarity between probe and target may range, sharing at least about50%, about 70%, and even about 85% sequence similarity.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. Generally, stringent wash temperature conditions areselected to be about 5° C. to about 2° C. lower than the melting point(Tm) for the specific sequence at a defined ionic strength and pH. Themelting point, or denaturation, of DNA occurs over a narrow temperaturerange and represents the disruption of the double helix into itscomplementary single strands. The process is described by thetemperature of the midpoint of transition, Tm, which is also called themelting temperature. Formulas are available in the art for thedetermination of melting temperatures.

Preferred hybridization conditions for the nucleotide sequence of theinvention include hybridization at 42° C. in 50% (w/v) formamide, 6×SSC,0.5% (w/v) SDS, 100 (g/ml salmon sperm DNA. Exemplary low stringencywashing conditions include hybridization at 42° C. in a solution of2×SSC, 0.5% (w/v) SDS for 30 minutes and repeating. Exemplary moderatestringency conditions include a wash in 2×SSC, 0.5% (w/v) SDS at 50° C.for 30 minutes and repeating. Exemplary high stringency conditionsinclude a wash in 0.1×SSC, 0.1% (w/v) SDS, at 65° C. for 30 minutes toone hour and repeating. Sequences that correspond to the promoter of thepresent invention may be obtained using all the above conditions. Forpurposes of defining the invention, the high stringency conditions areused.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, or 100nucleotides in length, or longer. Those of skill in the art understandthat to avoid a high similarity to a reference sequence due to inclusionof gaps in the polynucleotide sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of aligning sequences for comparison are well-known in the art.Thus, the determination of percent sequence identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4: 11-17; the local alignmentalgorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the globalalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local-alignment-method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85: 2444-2448; the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153;Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al.(1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller(1988) supra. A PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. The BLAST programs of Altschul et al (1990) J.Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul(1990) supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25: 3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3 and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2; and theBLOSUM62 scoring matrix or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizesthe number of matches and minimizes the number of gaps. GAP considersall possible alignments and gap positions and creates the alignment withthe largest number of matched bases and the fewest gaps. It allows forthe provision of a gap creation penalty and a gap extension penalty inunits of matched bases. GAP must make a profit of gap creation penaltynumber of matches for each gap it inserts. If a gap extension penaltygreater than zero is chosen, GAP must, in addition, make a profit foreach gap inserted of the length of the gap times the gap extensionpenalty. Default gap creation penalty values and gap extension penaltyvalues in Version 10 of the GCG Wisconsin Genetics Software Package forprotein sequences are 8 and 2, respectively. For nucleotide sequencesthe default gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

Identity to the sequence of the present invention would mean apolynucleotide sequence having at least 65% sequence identity, morepreferably at least 70% sequence identity, more preferably at least 75%sequence identity, more preferably at least 80% identity, morepreferably at least 85% sequence identity, more preferably at least 90%sequence identity and most preferably at least 95% sequence identity.

Promoter regions can be readily identified by one skilled in the art.The putative start codon containing the ATG motif is identified andupstream from the start codon is the presumptive promoter. By “promoter”is intended a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particular codingsequence. A promoter can additionally comprise other recognitionsequences generally positioned upstream or 5′ to the TATA box, referredto as upstream promoter elements, which influence the transcriptioninitiation rate. It is recognized that having identified the nucleotidesequences for the promoter region disclosed herein, it is within thestate of the art to isolate and identify further regulatory elements inthe region upstream of the TATA box from the particular promoter regionidentified herein. Thus the promoter region disclosed herein isgenerally further defined by comprising upstream regulatory elementssuch as those responsible for tissue and temporal expression of thecoding sequence, enhancers and the like. In the same manner, thepromoter elements which enable expression in the desired tissue such asmale tissue can be identified, isolated, and used with other corepromoters to confirm male tissue-preferred expression. By core promoteris meant the minimal sequence required to initiate transcription, suchas the sequence called the TATA box which is common to promoters ingenes encoding proteins. Thus the upstream promoter of Ms26 canoptionally be used in conjunction with its own or core promoters fromother sources. the promoter may be native or non-native to the cell inwhich it is found.

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of the heterologousnucleotide sequence. Less than the entire promoter region can beutilized and the ability to drive anther-preferred expression retained.However, it is recognized that expression levels of mRNA can bedecreased with deletions of portions of the promoter sequence. Thus, thepromoter can be modified to be a weak or strong promoter. Generally, by“weak promoter” is intended a promoter that drives expression of acoding sequence at a low level. By “low level” is intended levels ofabout 1/10,000 transcripts to about 1/100,000 transcripts to about1/500,000 transcripts. Conversely, a strong promoter drives expressionof a coding sequence at a high level, or at about 1/10 transcripts toabout 1/100 transcripts to about 1/1,000 transcripts. Generally, atleast about 30 nucleotides of an isolated promoter sequence will be usedto drive expression of a nucleotide sequence. It is recognized that toincrease transcription levels, enhancers can be utilized in combinationwith the promoter regions of the invention. Enhancers are nucleotidesequences that act to increase the expression of a promoter region.Enhancers are known in the art and include the SV40 enhancer region, the35S enhancer element, and the like.

The promoter of the present invention can be isolated from the 5′ regionof its native coding region of 5′ untranslation region (5′UTR). Likewisethe terminator can be isolated from the 3′ region flanking itsrespective stop codon. The term “isolated” refers to material such as anucleic acid or protein which is substantially or essentially free fromcomponents which normally accompany or interact with the material asfound in it naturally occurring environment or if the material is in itsnatural environment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in a cell otherthan the locus native to the material. Methods for isolation of promoterregions are well known in the art.

“Functional variants” of the regulatory sequences are also encompassedby the compositions of the present invention. Functional variantsinclude, for example, the native regulatory sequences of the inventionhaving one or more nucleotide substitutions, deletions or insertions.Functional variants of the invention may be created by site-directedmutagenesis, induced mutation, or may occur as allelic variants(polymorphisms).

As used herein, a “functional fragment” is a regulatory sequence variantformed by one or more deletions from a larger regulatory element. Forexample, the 5′ portion of a promoter up to the TATA box near thetranscription start site can be deleted without abolishing promoteractivity, as described by Opsahl-Sorteberg, H-G. et al., “Identificationof a 49-bp fragment of the HvLTP2 promoter directing aleruone cellspecific expression” Gene 341:49-58 (2004). Such variants should retainpromoter activity, particularly the ability to drive expression in maletissues. Activity can be measured by Northern blot analysis, reporteractivity measurements when using transcriptional fusions, and the like.See, for example, Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.), herein incorporated by reference.

Functional fragments can be obtained by use of restriction enzymes tocleave the naturally occurring regulatory element nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring DNA sequence; or can be obtained through the use ofPCR technology See particularly, Mullis et al. (1987) Methods Enzymol.155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, NewYork).

Sequences which hybridize to the regulatory sequences of the presentinvention are within the scope of the invention. Sequences thatcorrespond to the promoter sequences of the present invention andhybridize to the promoter sequences disclosed herein will be at least50% homologous, 70% homologous, and even 85% homologous or more with thedisclosed sequence.

Smaller fragments may yet contain the regulatory properties of thepromoter so identified and deletion analysis is one method ofidentifying essential regions. Deletion analysis can occur from both the5′ and 3′ ends of the regulatory region. Fragments can be obtained bysite-directed mutagenesis, mutagenesis using the polymerase chainreaction and the like. (See, Directed Mutagenesis: A Practical ApproachIRL Press (1991)). The 3′ deletions can delineate the essential regionand identify the 3′ end so that this region may then be operably linkedto a core promoter of choice. Once the essential region is identified,transcription of an exogenous gene may be controlled by the essentialregion plus a core promoter. By core promoter is meant the sequencecalled the TATA box which is common to promoters in all genes encodingproteins. Thus the upstream promoter of Ms26 can optionally be used inconjunction with its own or core promoters from other sources. Thepromoter may be native or non-native to the cell in which it is found.

The core promoter can be any one of known core promoters such as theCauliflower Mosaic Virus 35S or 19S promoter (U.S. Pat. No. 5,352,605),ubiquitin promoter (U.S. Pat. No. 5,510,474) the IN2 core promoter (U.S.Pat. No. 5,364,780) or a Figwort Mosaic Virus promoter (Gruber, et al.“Vectors for Plant Transformation” Methods in Plant Molecular Biologyand Biotechnology) et al. eds, CRC Press pp. 89-119 (1993)).

The regulatory region of Ms26 has been identified as including the 1005bp region upstream of the putative TATA box. See FIG. 7. Further, usingthe procedures outlined above, it has been determined that an essentialregion of the promoter includes the −180 bp upstream of the TATA box andspecifically, the −176 to −44 region is particularly essential.

Promoter sequences from other plants may be isolated according towell-known techniques based on their sequence homology to the promotersequence set forth herein. In these techniques, all or part of the knownpromoter sequence is used as a probe which selectively hybridizes toother sequences present in a population of cloned genomic DNA fragments(i.e. genomic libraries) from a chosen organism. Methods are readilyavailable in the art for the hybridization of nucleic acid sequences.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least aboutnucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see e.g. Innis et al., eds., (1990) PCR Protocols,A Guide to Methods and Applications, Academic Press).

Further, a promoter of the present invention can be linked withnucleotide sequences other than the Ms26 gene to express otherheterologous nucleotide sequences. The nucleotide sequence for thepromoter of the invention, as well as fragments and variants thereof,can be provided in expression cassettes along with heterologousnucleotide sequences for expression in the plant of interest, moreparticularly in the male tissue of the plant. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the nucleotide sequence to be under the transcriptional regulation ofthe promoter. These expression cassettes are useful in the geneticmanipulation of any plant to achieve a desired phenotypic response.Examples of other nucleotide sequences which can be used as theexogenous gene of the expression vector with the Ms26 promoter includecomplementary nucleotidic units such as antisense molecules (callaseantisense RNA, bamase antisense RNA and chalcone synthase antisense RNA,Ms45 antisense RNA), ribozymes and external guide sequences, an aptameror single stranded nucleotides. The exogenous nucleotide sequence canalso encode auxins, rol B, cytotoxins, diptheria toxin, DAM methylase,avidin, or may be selected from a prokaryotic regulatory system. By wayof example, Mariani, et al., Nature; Vol. 347; pp. 737; (1990), haveshown that expression in the tapetum of either Aspergillus oryzaeRNase-T1 or an RNase of Bacillus amyloliquefaciens, designated“barnase,” induced destruction of the tapetal cells, resulting in maleinfertility. Quaas, et al., Eur. J. Biochem. Vol. 173: pp. 617 (1988),describe the chemical synthesis of the RNase-T1, while the nucleotidesequence of the barnase gene is disclosed in Hartley, J. Molec. Biol.;Vol. 202: pp. 913 (1988). The rolB gene of Agrobacterium rhizogenescodes for an enzyme that interferes with auxin metabolism by catalyzingthe release of free indoles from indoxyl-β-glucosides. Estruch, et al.,EMBO J. Vol. 11: pp. 3125 (1991) and Spena, et al., Theor. Appl. Genet.;Vol. 84: pp. 520 (1992), have shown that the anther-specific expressionof the rolB gene in tobacco resulted in plants having shriveled anthersin which pollen production was severely decreased and the rolB gene isan example of a gene that is useful for the control of pollenproduction. Slightom, et al., J. Biol. Chem. Vol. 261: pp. 108 (1985),disclose the nucleotide sequence of the rolB gene. DNA moleculesencoding the diphtheria toxin gene can be obtained from the AmericanType Culture Collection (Rockville, Md.), ATCC No. 39359 or ATCC No.67011 and see Fabijanski, et al., E.P. Appl. No. 90902754.2, “MolecularMethods of Hybrid Seed Production” for examples and methods of use. TheDAM methylase gene is used to cause sterility in the methods discussedat U.S. Pat. No. 5,689,049 and PCT/US95/15229 Cigan, A. M. andAlbertsen, M. C., “Reversible Nuclear Genetic System for Male Sterilityin Transgenic Plants”. Also see discussion of use of the avidin gene tocause sterility at U.S. Pat. No. 5,962,769 “Induction of Male Sterilityin Plants by Expression of High Levels of Avidin” by Albertsen et al.

The invention includes vectors with the Ms26 gene. A vector is preparedcomprising Ms26, a promoter that will drive expression of the gene inthe plant and a terminator region. As noted, the promoter in theconstruct may be the native promoter or a substituted promoter whichwill provide expression in the plant. Selection of the promoter willdepend upon the use intended of the gene. The promoter in the constructmay be an inducible promoter, so that expression of the sense orantisense molecule in the construct can be controlled by exposure to theinducer.

Other components of the vector may be included, also depending uponintended use of the gene. Examples include selectable markers, targetingor regulatory sequences, stabilizing or leader sequences, etc. Generaldescriptions and examples of plant expression vectors and reporter genescan be found in Gruber, et al., “Vectors for Plant Transformation” inMethod in Plant Molecular Biology and Biotechnology, Glick et al eds;CRC Press pp. 89-119 (1993). The selection of an appropriate expressionvector will depend upon the host and the method of introducing theexpression vector into the host. The expression cassette will alsoinclude at the 3′ terminus of the heterologous nucleotide sequence ofinterest, a transcriptional and translational termination regionfunctional in plants. The termination region can be native with thepromoter nucleotide sequence of the present invention, can be nativewith the DNA sequence of interest, or can be derived from anothersource. Convenient termination regions are available from the Ti-plasmidof A. tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau et al. Mol. Gen. Genet.262:141-144 (1991); Proudfoot, Cell 64:671-674 (1991); Sanfacon et al.Genes Dev. 5:141-149 (1991); Mogen et al. Plant Cell 2:1261-1272 (1990);Munroe et al. Gene 91:151-158 (1990); Ballas et al. Nucleic Acids Res.17:7891-7903 (1989); Joshi et al. Nucleic Acid Res. 15:9627-9639 (1987).

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein et al. Proc. Nat. Acad. Sci. USA 86:6126-6130 (1989);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allisonet al.; MDMV leader (Maize Dwarf Mosaic Virus), Virology 154:9-20(1986); human immunoglobulin heavy-chain binding protein (BiP), Macejaket al. Nature 353:90-94 (1991); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al. Nature325:622-625 (1987); Tobacco mosaic virus leader (TMV), Gallie et al.(1989) Molecular Biology of RNA, pages 237-256; and maize chloroticmottle virus leader (MCMV) Lommel et al. Virology 81:382-385 (1991). Seealso Della-Cioppa et al. Plant Physiology 84:965-968 (1987). Thecassette can also contain sequences that enhance translation and/or mRNAstability such as introns.

In those instances where it is desirable to have the expressed productof the heterologous nucleotide sequence directed to a particularorganelle, particularly the plastid, amyloplast, or to the endoplasmicreticulum, or secreted at the cell's surface or extracellularly, theexpression cassette can further comprise a coding sequence for a transitpeptide. Such transit peptides are well known in the art and include,but are not limited to, the transit peptide for the acyl carrierprotein, the small subunit of RUBISCO, plant EPSP synthase, and thelike. One skilled in the art will readily appreciate the many optionsavailable in expressing a product to a particular organelle. Forexample, the barley alpha amylase sequence is often used to directexpression to the endoplasmic reticulum (Rogers, J. Biol. Chem.260:3731-3738 (1985)). Use of transit peptides is well known (e.g., seeU.S. Pat. Nos. 5,717,084; 5,728,925).

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions, such astransitions and transversions, can be involved.

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the promoter. Ingeneral, the vectors should be functional in plant cells. At times, itmay be preferable to have vectors that are functional in E. coli (e.g.,production of protein for raising antibodies, DNA sequence analysis,construction of inserts, obtaining quantities of nucleic acids). Vectorsand procedures for cloning and expression in E. coli are discussed inSambrook et al. (supra).

The transformation vector comprising the promoter sequence of thepresent invention operably linked to a heterologous nucleotide sequencein an expression cassette, can also contain at least one additionalnucleotide sequence for a gene to be cotransformed into the organism.Alternatively, the additional sequence(s) can be provided on anothertransformation vector.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample, Jefferson et al. (1991) in Plant Molecular Biology Manual, ed.Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al. Mol.Cell. Biol. 7:725-737 (1987); Goff et al. EMBO J. 9:2517-2522 (1990);Kain et al. BioTechniques 19:650-655 (1995); and Chiu et al. CurrentBiology 6:325-330 (1996).

Selectable marker genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to,genes encoding resistance to chloramphenicol, Herrera Estrella et al.EMBO J. 2:987-992 (1983); methotrexate, Herrera Estrella et al. Nature303:209-213 (1983); Meijer et al. Plant Mol. Biol. 16:807-820 (1991);hygromycin, Waldron et al. Plant Mol. Biol. 5:103-108 (1985); Zhijian etal. Plant Science 108:219-227 (1995); streptomycin, Jones et al. Mol.Gen. Genet. 210:86-91 (1987); spectinomycin, Bretagne-Sagnard et al.Transgenic Res. 5:131-137 (1996); bleomycin, Hille et al. Plant Mol.Biol. 7:171-176 (1990); sulfonamide, Guerineau et al. Plant Mol. Biol.15:127-136 (1990); bromoxynil, Stalker et al. Science 242:419-423(1988); glyphosate, Shaw et al. Science 233:478-481 (1986);phosphinothricin, DeBlock et al. EMBO J. 6:2513-2518 (1987).

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription or transcript andtranslation of the sequence to effect phenotypic changes in theorganism. Thus, any method which provides for efficienttransformation/transfection may be employed.

Methods for introducing expression vectors into plant tissue availableto one skilled in the art are varied and will depend on the plantselected. Procedures for transforming a wide variety of plant speciesare well known and described throughout the literature. See, forexample, Miki et al, “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biotechnology, supra; Klein et al,Bio/Technology 10:268 (1992); and Weising et al., Ann. Rev. Genet. 22:421-477 (1988). For example, the DNA construct may be introduced intothe genomic DNA of the plant cell using techniques such asmicroprojectile-mediated delivery, Klein et al., Nature 327: 70-73(1987); electroporation, Fromm et al., Proc. Natl. Acad. Sci. 82: 5824(1985); polyethylene glycol (PEG) precipitation, Paszkowski et al., EMBOJ. 3: 2717-2722 (1984); direct gene transfer WO 85/01856 and EP No. 0275 069; in vitro protoplast transformation U.S. Pat. No. 4,684,611; andmicroinjection of plant cell protoplasts or embryogenic callus.Crossway, Mol. Gen. Genetics 202:179-185 (1985). Co-cultivation of planttissue with Agrobacterium tumefaciens is another option, where the DNAconstructs are placed into a binary vector system. See e.g., U.S. Pat.No. 5,591,616; Ishida et al., “High Efficiency Transformation of Maize(Zea mays L.) mediated by Agrobacterium tumefaciens” NatureBiotechnology 14:745-750 (1996). The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct into the plant cell DNA when the cell is infected by thebacteria. See, for example Horsch et al., Science 233: 496-498 (1984),and Fraley et al., Proc. Natl. Acad. Sci. 80: 4803 (1983).

Standard methods for transformation of canola are described at Moloneyet al. “High Efficiency Transformation of Brassica napus usingAgrobacterium Vectors” Plant Cell Reports 8:238-242 (1989). Corntransformation is described by Fromm et al, Bio/Technology 8:833 (1990)and Gordon-Kamm et al, supra. Agrobacterium is primarily used in dicots,but certain monocots such as maize can be transformed by Agrobacterium.See supra and U.S. Pat. No. 5,550,318. Rice transformation is describedby Hiei et al., “Efficient Transformation of Rice (Oryza sativs L.)Mediated by Agrobacterium and Sequence Analysis of the Boundaries of theT-DNA” The Plant Journal 6(2): 271-282 (1994, Christou et al, Trends inBiotechnology 10:239 (1992) and Lee et al, Proc. Nat'l Acad. Sci. USA88:6389 (1991). Wheat can be transformed by techniques similar to thoseused for transforming corn or rice. Sorghum transformation is describedat Casas et al, supra and sorghum by Wan et al, Plant Physicol. 104:37(1994). Soybean transformation is described in a number of publications,including U.S. Pat. No. 5,015,580.

Further detailed description is provided below by way of instruction andillustration and is not intended to limit the scope of the invention.

EXAMPLE 1 Identification and Cosegregation of Ms26-M2::Mu8

Families of plants from a Mutator (Mu) population were identified thatsegregated for plants that were mostly male sterile, with none or only afew extruded abnormal anthers, none of which had pollen present. Malesterility is expected to result from those instances where a Mu elementhas randomly integrated into a gene responsible for some step inmicrosporogenesis, disrupting its expression. Plants from a segregatingF₂ family in which the male sterile mutation was designatedms26*-SBMu200, were grown and classified for male fertility/sterilitybased on the above criteria. Leaf samples were taken and DNAsubsequently isolated on approximately 20 plants per phenotypicclassification, that is male fertility vs. male sterility.

Southern analysis was performed to confirm association of Mu withsterility. Southern analysis is a well known technique to those skilledin the art. This common procedure involves isolating the plant DNA,cutting with restriction endonucleases, fractioning the cut DNA bymolecular weight on an agarose gel, and transferring to nylon membranesto fix the separated DNA. These membranes are subsequently hybridizedwith a probe fragment that was radioactively labeled with P³²P-dCTP, andwashed in an SDS solution. Southern, E., “Detection of SpecificSequences Among DNA Fragments by Gel Electrophoresis,” J. Mol. Biol.98:503-317 (1975). Plants from a segregating F₂ ms26*-SBMu200 familywere grown and classified for male fertility/sterility. Leaf samples andsubsequent DNA isolation was conducted on approximately 20 plants perphenotypic classification. DNA (˜7 ug) from 5 fertile and 12 sterileplants was digested with EcoRI and electrophoresed through a 0.75%agarose gel. The digested DNA was transferred to nylon membrane viaSouthern transfer. The membrane was hybridized with an internal fragmentfrom the Mu8 transposon. Autoradiography of the membrane revealedcosegregation of a 5.5 Kb EcoRI fragment with the sterility phenotype asshown in FIG. 1. This EcoRI band segregated in the fertile plantssuggesting a heterozygous wild type condition for the allele

EXAMPLE 2 Library Construction, Screening, and Mapping

The process of genomic library screenings is commonly known among thoseskilled in the art and is described at Sambrook, J., Fritsch, E. F.,Maniatis T., et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor Lab Press, Plainview, N.Y. (1989).Libraries were created as follows.

DNA from a sterile plant was digested with EcoRI and run on apreparative gel. DNA with a molecular weight between 5.0 and 6.0 Kb wasexcised from the gel, electroeluted and ethanol precipitated. This DNAwas ligated into the Lambda Zap vector (Stratagene™) using themanufacturer's protocol. The ligated DNA was packaged into phageparticles using Gigapack Gold (Stratagene™). Approximately 500,000 PFUwere plated and lifted onto nitrocellulose membranes. Membranes werehybridized with the Mu8 probe. A pure clone was obtained after 3 roundsof screening. The insert was excised from the phage as a plasmid anddesignated SBMu200-3.1. A PstI border fragment from this clone wasisolated and used to reprobe the orginal EcoRI cosegregation blot asshown in FIG. 2B. The 5.5 kb EcoRI fragment is homozygous in all thesterile plants, which confirms that the correct Mu fragment wasisolated. Three of the fertile plants are heterozygous for the 5.5 kbEcoRI band and a 4.3 Kb EcoRI band. Two of the fertile plants arehomozygous for the 4.3 kb EcoRI band, presumably the wild type allele.

The PstI probe was used to map the ms*-SBMu200 mutation in an RFLPmapping population. The mutant mapped to the short arm of chromosome 1,near the male sterile locus, Ms26 (Loukides et al., (1995) Amer. J. Bot82, 1017-1023). To test whether ms*-SBMu200 was an allele of ms26-ref,ms*-SBMu200 and ms26-ref were crossed with each other using a knownheterozygote as the pollen donor. The testcross progeny segregatedmale-sterile and wild-type plants in a 1:1 ratio, indicating allelismbetween ms*-SBMu200 and ms26-ref. The ms*-SBMu200 allele was designatedms26-m2::Mu8. The map location is shown in FIG. 12.

EXAMPLE 3 Identification and Cloning of Additional Ms26 Alleles

Three additional Mu insertion mutations in Ms26 were identified by usinga polymerase chain reaction (PCR) primer for Mu and a gene specificprimer for Ms26. Sequence analyses of the PCR products showed that allthree Mu insertions occurred in the second exon (FIG. 1). The F₂ seedsfrom one of these families were grown and examined for malefertility/sterility. Southern blot analyses of this family confirmed thecosegregation of the Mu insertion in Ms26 with the male-sterilephenotype.

The ms26 allele described in Loukides et al., (1995) Amer. J. Bot 82,1017-1023 and designated ms26-ref was also investigated. To analyze themutation in ms26-ref, Ms26 genomic sequences were cloned from ms26-refsterile and fertile plants. Ms26 was cloned as a ˜4.2 kb EcoRI fragmentand ms26-ref cloned as a ˜6 kb HindIII fragment and an overlapping ˜2.3kb EcoRI fragment from the sterile plant. Sequence analysis revealed thepresence of a new segment (1,430 bp) in the last exon of the ms26-refallele shown in FIG. 1. An 8 bp host site duplication (GCCGGAGC) wasfound that flanks the inserted element and the element also contains a15 bp terminal inverted repeat (TIR) (TAGGGGTGAAAACGG; SEQ ID NO: 23).The transposon sequence is shown in SEQ ID NO: 10. The ms26-ref genomicsequence in its entirety is shown in SEQ ID NO: 11. A variant of thems26-ref allele was also found. Sequence analysis of this allele,designated ms26′-0406, was found to have lost the 1430 bp segment foundin the last exon of the ms26-ref allele but left an 8 bp footprint atthe site of insertion. Plants homozygous for the ms26′-0406 allele weremale sterile. A comparison of the excision allele, ms26′-0406 (SEQ IDNO: 8) with the region in the wild-type Ms26 gene (SEQ ID NO: 9) isshown in FIG. 13.

EXAMPLE 4 Expression Analysis and cDNA Isolation

Northern analysis can be used to detect expression of genescharacteristic of anther development at various states ofmicrosporogenesis. Northern analysis is also a commonly used techniqueknown to those skilled in the art and is similar to Southern analysisexcept that mRNA rather than DNA is isolated and placed on the gel. TheRNA is then hybridzed with the labeled probe. Potter, E., et al.,“Thyrotrotropin Releasing Hormone Exerts Rapid Nuclear Effects toIncrease Production of the Primary Prolactin in RNA Transcript,” Proc.Nat. Acad. Sci. USA 78:6662-6666 (1981), Lechelt, et al., “Isolation &Molecular Analysis of the Plows,” Mol. Gen. Genet. 219:225-234 (1989).The PstI fragment from the SBMu200-3.1 clone was used to probe aNorthern blot containing kernel, immature ear, seedling and tassel RNA.A signal was seen only in tassel RNA at approximately the quartet stageof microsporogenesis, as reflected in FIG. 3. The transcript is about2.3 kb in length. The same probe was also used to screen a cDNA libraryconstructed from mRNA isolated from meiotic to late uninucleate stagedanthers. One clone, designated Ms26-8.1, was isolated from the library.

EXAMPLE 5 Sequence and Expression Analysis

The SBMu200-3.1 genomic clone and the Ms26-8.1 cDNA clone were sequencedby Loftstrand Labs Limited. Sanger, F., Nicklen, S., Coulson A. R.(1977) “DNA sequencing with chain terminating inhibitors” Proc. Natl.Acad. Sci. USA 74:5463-5467. The sequences are set forth in FIG. 4 andthe comparison is at FIG. 5. The cDNA/genomic comparison reveals fiveintrons are present in the genomic clone. The Mu8 insertion occurs inexon 1. Testing for codon preference and non-randomness in the thirdposition of each codon was consistent with the major ORF in the cDNAbeing the likely protein-coding ORF. There is a putative Met start codonat position 1089 in the genomic clone. The cDNA homology with respect tothe genomic clone begins at nucleotide 1094. Thus Ms26-8.1 does notrepresent a full length clone and lacks 5 bases up to the putative Metstart codon. A database search revealed significant homology to P450enzymes found in yeast, plants and mammals. P450 enzymes have beenwidely studied and three characteristic protein domains have beenelucidated. The Ms26 protein contains several structural motifscharacteristic of eukaryotic P450's, including the heme-binding domainFxxGxRxCxG (domain D; SEQ ID NO: 24), domain A A/GGXD/ETT/S(dioxygen-binding), domain B (steroid-binding), and domain C. The highlyconserved heme-binding motif was found in MS26 as FQAGPRICLG (SEQ ID NO:25), 51 amino acids away from C-terminus. The dioxygen binding domainAGRDTT (SEQ ID NO: 26) was located between amino acids 320-325. Thesteroid-binding domain was found as LVYLHACVTETLR (SEQ ID NO: 27), aminoacids 397-409. The most significant homologous sequence detected inGenebank database is a deduced protein sequence from rice (GeneBankaccession number 19071651). The second highest homologous sequence is aputative Arabidopsis P450 gene (CYP704B1) whose function is alsounknown. FIG. 14A shows a sequence alignment between CYP704B1 (SEQ IDNO: 12) and Ms26 (SEQ ID NO: 13). Phylogenetic tree analysis of someP450 genes revealed that Ms26 is most closely related to P450s involvedin fatty acid omega-hydroxylation found in Arabidopsis thaliana andVicia sativa (FIG. 14B). The translational frame shift caused in thems26′-0406 excision mutation is believed to destroy the activity of theheme binding domain, thus resulting in sterility. See the comparison atFIG. 15 (Ms26 cDNA at SEQ ID NO: 14; fertile exon 5 region at SEQ ID NO:15 and sterile exon 5 region is SEQ ID NO: 16).

Further expression studies were done using the Ms26 cDNA probe against anorthern containing mRNA at discrete stages of microsporogenesis. FIG.6A shows a Northern blot with RNA samples from different tissuesincluding root (1), leaf (2), husk (3), cob (4), ear spikelet (5), silk(6), immature embryo (7) mature embryo (8), and tassel from, fertileplant (9), ms26-m2::Mu8 sterile plant (10), ms26-ref sterile plant (11)and fertile plant (12). A hybridization signal using Ms26 cDNA wasdetected only in tassel tissues. FIG. 6B shows a Northern blotcontaining mRNA at discrete stages of microsporogenesis. Hybridizationsignals using Ms26 cDNA were detected from meiosis II/quartet stage (4)to late-uninucleate stage (10), with the maximal signal being observedfrom early-uninucleate through late-uninucleate stage (10).

EXAMPLE 6 Identification of Promoter and its Essential Regions

A putative TATA box can be identified by primer extension analysis asdescribed in by Current Protocols in Molecular Biology, Ausubel, F. M.et al. eds; John Wiley and Sons, New York pp. 4.8.1-4.8.5 (1987).

Regulatory regions of anther genes, such as promoters, may be identifiedin genomic subclones using functional analysis, usually verified by theobservation of reporter gene expression in anther tissue and a lowerlevel or absence of reporter gene expression in non-anther tissue. Thepossibility of the regulatory regions residing “upstream” or 5′ ward ofthe translational start site can be tested by subcloning a DNA fragmentthat contains the upstream region into expression vectors for transientexpression experiments. It is expected that smaller subgenomic fragmentsmay contain the regions essential for male-tissue preferred expression.For example, the essential regions of the CaMV 19S and 35S promotershave been identified in relatively small fragments derived from largergenomic pieces as described in U.S. Pat. No. 5,352,605.

The selection of an appropriate expression vector with which to test forfunctional expression will depend upon the host and the method ofintroducing the expression vector into the host and such methods arewell known to one skilled in the art. For eukaryotes, the regions in thevector include regions that control initiation of transcription andcontrol processing. These regions are operably linked to a reporter genesuch as UidA, encoding β -glucuronidase (GUS), or luciferase. Generaldescriptions and examples of plant expression vectors and reporter genescan be found in Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology; Glick, et al. eds;CRC Press; pp. 89-119; (1993). GUS expression vectors and GUS genecassettes are commercially available from Clonetech, Palo Alto, Calif.,while luciferase expression vectors and luciferase gene cassettes areavailable from Promega Corporation, Madison, Wis. Ti plasmids and otherAgrobacterium vectors are described in Ishida, Y., et al., NatureBiotechnology; Vol. 14; pp. 745-750; (1996) and in U.S. Pat. No.5,591,616 “Method for Transforming Monocotyledons” (1994).

Expression vectors containing putative regulatory regions located ingenomic fragments can be introduced into intact tissues such as stagedanthers, embryos or into callus. Methods of DNA delivery includemicroprojectile bombardment, DNA injection, electroporation andAgrobacterium-mediated gene transfer (see Gruber, et al., “Vectors forPlant Transformation,” in Methods in Plant Molecular Biology andBiotechnology, Glick, et al. eds.; CRC Press; (1993); U.S. Pat. No.5,591,616; and Ishida, Y., et al., Nature Biotechnology; Vol. 14; pp.745-750; (1996)). General methods of culturing plant tissues are foundin Gruber, et al., supra and Glick, supra.

For the transient assay system, staged, isolated anthers are immediatelyplaced onto tassel culture medium (Pareddy, D. R. and J. F. Petelino,Crop Sci. J.; Vol. 29; pp. 1564-1566; (1989)) solidified with 0.5%Phytagel (Sigma, St. Louis) or other solidifying media. The expressionvector DNA is introduced within 5 hours preferably bymicroprojectile-mediated delivery with 1.2 μm particles at 100-1100 Psi.After DNA delivery, the anthers are incubated at 26° C. upon the sametassel culture medium for 17 hours and analyzed by preparing a wholetissue homogenate and assaying for GUS or for lucifierase activity (seeGruber, et al., supra).

Upstream of the likely translational start codon of Ms26, 1088 bp of DNAwas present in the genomic clone ms26-m2::Mu8. Translational fusions viaan engineered NcoI site were generated with reporter genes encodingluciferase and β-glucuronidase to test whether this fragment of DNA hadpromoter activity in transient expression assays of bombarded planttissues. Activity was demonstrated in anthers and not in coleoptiles,roots and calli, suggesting anther-preferred or anther-specific promoteractivity.

A reasonable TATA box was observed by inspection, about 83-77 bpupstream of the translational start codon. The genomic clonems26-m2::Mu8 thus includes about 1005 bp upstream of the possible TATAbox. For typical plant genes, the start of transcription is 26-36 bpdownstream of the TATA box, which would give the Ms26 mRNA a5′-nontranslated leader of about 48-58 nt. The total ms26-m2::Mu8subgenomic fragment of 1088 bp, including nontranslated leader, start oftranscription, TATA box and sequences upstream of the TATA box, was thusshown to be sufficient for promoter activity. See SEQ. ID NO.5. Theputative TATA box (TATATCA) is underlined. Thus, the present inventionencompasses a DNA molecule having a nucleotide sequence of SEQ ID NO: 5(or those with sequence identity) and having the function of a maletissue-preferred regulatory region.

Deletion analysis can occur from both the 5′ and 3′ ends of theregulatory region: fragments can be obtained by site-directedmutagenesis, mutagenesis using the polymerase chain reaction, and thelike (Directed Mutagenesis: A Practical Approach; IRL Press; (1991)).The 3′ end of the male tissue-preferred regulatory region can bedelineated by proximity to the putative TATA box or by 3′ deletions ifnecessary. The essential region may then be operably linked to a corepromoter of choice. Once the essential region is identified,transcription of an exogenous gene may be controlled by the maletissue-preferred region of Ms26 plus a core promoter. The core promotercan be any one of known core promoters such as a Cauliflower MosaicVirus 35S or 19S promoter (U.S. Pat. No. 5,352,605), Ubiquitin (U.S.Pat. No. 5,510,474), the IN2 core promoter (U.S. Pat. No. 5,364,780), ora Figwort Mosaic Virus promoter (Gruber, et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology;Glick, et al. eds.; CRC Press; pp. 89-119; (1993)). Preferably, thepromoter is the core promoter of a male tissue-preferred gene or theCaMV 35S core promoter. More preferably, the promoter is a promoter of amale tissue-preferred gene and in particular, the Ms26 core promoter.

Further mutational analysis, for example by linker scanning, a methodwell known to the art, can identify small segments containing sequencesrequired for anther-preferred expression. These mutations may introducemodifications of functionality such as in the levels of expression, inthe timing of expression, or in the tissue of expression. Mutations mayalso be silent and have no observable effect.

The foregoing procedures were used to identify essential regions of theMs26 promoter. After linking the promoter with the luciferase markergene deletion analysis was performed on the regions of the promoterupstream of the putative TATA box, as represented in FIG. 8. The x-axisof the bar graph indicates the number of base pairs immediately upstreamof the putative TATA box retained in a series of deletion derivativesstarting from the 5′ end of the promoter. The y-axis shows thenormalized luciferase activity as a percent of full-length promoteractivity.

As is evident from the graph, approximately 176 bp immediately upstreamof the TATA box was sufficient, when coupled to the core promoter(putative TATA box through start of transcription), plus 5′nontranslated leader, for transient expression in anthers. By contrast,luciferase activity was minimal upon further deletion from the 5′ end to91 bp upstream of the putative TATA box. This 176 bp upstream of theputative TATA box through the nontranslated leader can be considered aminimal promoter, which is further represented at FIG. 9. The TATA boxis underlined. Deletion within the full-length promoter from −176through −92 relative to the TATA box reduced activity to about 1% ofwild type. Deletion of −39 through −8 did not greatly reduce activity.Therefore the −176 to −44 bp region contains an essential region andthus would constitute an upstream enhancer element conferring antherexpression on the promoter, which we refer to as an “anther box”.

Linker scanning analysis was conducted across the anther box in 9-10 bpincrements. The locations of the linker scanning substitutions in thisregion are shown in FIG. 9, and the expression levels of the mutantsrelative to the wild type sequence are shown in FIG. 10. The mostdrastic effect on transient expression in anthers was observed formutants LS12 and LS13, in the region 52-71 bp upstream of the putativeTATA box. A major effect on transient expression in anthers was alsoobserved for mutants LS06, LS07, LS08 and LS10, within the region 82-131bp upstream of the putative TATA box. Sequences within the anther boxrequired for wild type levels of transient expression in anthers arethus demonstrated in the −52 to −131 region relative to the putativeTATA box, particularly the −52 to −71 region.

EXAMPLE 7 Ms26 Sorghum, Rice and Maize Comparison

As noted above, Ms26 is a male fertility gene in maize. When it ismutated, and made homozygous recessive, male sterility will result. Anorthologue of Ms26 was identified in sorghum. The sorghum orthologue ofthe Ms26 cDNA was isolated by using the maize Ms26 gene primers in apolymerase chain reaction with sorghum tassel cDNA as the template. Theresultant cDNA fragment was sequenced by methods described supra andthen compared to the Ms26 cDNA from maize. Nucleotide sequencecomparisons are set forth in FIG. 11 and show 90% identity. Anorthologue from rice was also identified and the predicted codingsequence is SEQ ID NO: 17 and protein is SEQ ID NO: 18. It has oneintron less than the maize and sorghum Ms26, and the coding sequencesare highly conserved.

Identification of the sorghum and rice promoters was accomplished. FIG.16 shows an alignment of the Ms26 promoter of corn (SEQ ID NO: 5),sorghum (SEQ ID NO: 19) and rice (SEQ ID NO: 20). The last three basesof the corn promoter shown in the figure is the ATG start oftranslation.

Alignment as reflected in FIG. 17 of the maize Ms26 protein (SEQ ID NO:2), rice Ms26 protein(SEQ ID NO: 18) and sorghum Ms26 protein (SEQ IDNO: 4), and a consensus sequence (SEQ ID NO: 35). The comparison ofprotein sequences shows the protein is highly conserved among theorthologues, with the rice protein sharing 92% similarity and 86%identity when compared to the maize orthologue. The predicted tissuespecificity in rice and sorghum is further reflected in a comparison ofthe Ms26 protein in the sorghum and rice EST database derived frompanicle (flower) libraries. Sorghum sequences producing significantalignments (GenBank accession numbers B1075441.1; B1075273.1;B1246000.1; B1246162.1; BG948686.1; B1099541.1 and BG948366.1, amongothers) all were sequences from immature panicle of sorghum, andsequences showing significant alignment in rice (GenBank accessionnumbers C73892.1; CR290740.1, among others) were also from rice immaturepanicle.

As is evident from the above, nucleotide sequences which map to theshort arm of chromosome 1 of the Zea mays genome, at the same site asthe Ms26 gene, ms26-m2::Mu8 and its alleles, are genes critical to malefertility in plants, that is, are necessary for fertility of a plant,or, when mutated from the sequence found in a fertile plant, causesterility in the plant.

Thus it can be seen that the invention achieves at least all of itsobjectives.

1. An isolated polynucleotide comprising a sequence encoding the aminoacid sequence of SEQ ID NO: 2.